Identification of novel virulence determinants is a crucial goal of infectious disease research and necessary for a comprehensive understanding of the complex mechanisms that encompass microbial pathogenicity. This research is critical for the development of innovative treatment options in the era of increasing antibiotic resistance. Pseudomonas aeruginosa is a significant opportunistic pathogen and the cause of various nosocomial infections. Despite what is understood about P. aeruginosa pathogenesis, individual strains can substantially differ in their virulence and responsiveness to current treatment options. The specific factors responsible for much of this difference in virulence are currently unknown. A large portion of the P. aeruginosa genome varies between individual strains, and this variable accessory genome has been shown to play a role in the pathogenesis of P. aeruginosa. It is hypothesized that novel virulence factors within the P. aeruginosa accessory genome account for some of the significant differences in virulence between strains. The hypothesis will be tested by completing the following two specific aims: In Aim 1, the genome sequences of approximately 100 P. aeruginosa clinical bloodstream isolates will be determined utilizing next generation Illumina sequencing. A directed bioinformatic approach will be used to determine regions of the P. aeruginosa accessory genome that are significantly associated with highly virulent isolates, thus likely to harbor novel virulence factors. In Aim 2, piecemeal deletin of virulence-associated accessory genomic elements will be performed to identify the specific gene(s) responsible for virulence in a mouse bacteremia model. Upon completion of these specific aims, it is expected that an extensive set of novel virulence factors will be identified tat directly contribute to the pathogenesis of highly virulent P. aeruginosa strains. The information gained from the proposed experiments will lead to elucidation of the complex pathogenic mechanisms of P. aeruginosa. Additionally these factors could be used as specific biomarkers of highly virulent strains allowing clinicians to identify patients in need of particularly aggressve interventions. Moreover, this work will lay the foundation for future studies into the development of innovative therapeutic interventions.